Drosophila innate immunity suppresses the survival of xenografted mammalian tumor cells

Patient-derived xenograft (PDX) is an emerging tool established in immunodeficient vertebrate models to assess individualized treatments for cancer patients. Current xenograft models are deficient in adaptive immune systems. However, the precise role of the innate immunity in the xenograft models is unknown. With conserved signaling pathways and established genetic tools, Drosophila has contributed to the understanding of the mechanism of tumor growth as well as tumor–host interactions for decades, making it a promising candidate model for studying whether or not the hosts’ innate immunity can accommodate transplanted human tumor cells. Here we show initial observations that assess the behavior and impact of several human tumor cell lines when transplanted into Drosophila. We found that some injected cell lines persisted for a longer duration and reduced hosts’ lifespan. In particular, the human lung cancer cell line A549 were observed adjacent to the fly host tissues. We examined two factors that affect the survivability of cancer cells: (1) the optimal temperature of each cell line and (2) the innate immunity of Drosophila hosts. Especially, transplanted human tumor cells survived longer in immunodeficient flies, suggesting that the host innate immune system impedes the growth of xenografted cells. Our attempts for xenografting fly models thus provide necessary steps to overcome for establishing PDX cancer models using invertebrates.

www.nature.com/scientificreports/ Despite many fruitful achievements in allograft models, however, xenografting experiments in Drosophila have not been established. This may be due to the extreme differences in the body structure, organ systems, and living conditions between vertebrates and Drosophila 5 . One factor that may impact the survival and growth of xenografted cells is the immune system of the hosts. Compared to vertebrates that have both acquired and innate immunity, the Drosophila immune system only consists of the innate immunity, which is regulated by the Toll and Imd pathways 12 . Various studies have shown the immune system's efficacy in removing bacteria and fungi through the production of antibacterial peptides, such as Drosomycin and Diptericin 13 . However, whether or not the innate immune system can protect the hosts from foreign cells derived from nonbacterial and nonfungal origins is unclear.
In this study, we performed xenograft experiments by injecting human tumor cell lines into adult Drosophila hosts. Among cell lines that we injected, A549 and DLD-1 cells survived for approximately 12 days within the control hosts, and these host flies exhibited significantly shortened life span. We further show that the human tumor cells remained longer and spread more widely when transplanted into immunodeficient host flies, which causes mortality of host flies. These results together suggest that the Drosophila innate immune system plays a role in negatively influencing human tumor cell growth, providing a caution for the influence of the innate immune system when generating PDX models.
The cell suspension was extracted into a glass needle (Drummond Scientific Company, Broomall, Pennsylvania, USA, Catalog No. 1-000-0300) and then injected into the abdomen of young adult female flies (within 5 days upon eclosion) using a NARISHIGE IM300 Microinjector (Narishige Scientific Instrument Lab., Tokyo, Japan) with the following settings: N 2 gas fill pressure at 20.0 PSI, injection pressure at 5.0 PSI, and balance pressure at 1.8 PSI. Injected host flies were incubated at 29 °C.
The cell suspension was injected into the young adult female abdomen as specified in the allograft protocol above. Fly hosts were w 1118 , Rel E20 , Dredd D44 , or PGRP-LC Δ5 as specified in the corresponding experiments in the result section. Injected host flies were incubated at 29 °C.
Drosophila life-span measurements. Flies were transferred to fresh food every day for 7 days following injection to avoid infection and every 2 days thereafter. Living flies were counted every 2 days. Lifespan analyses were performed, and log rank test statistics were calculated using the R version 4.0.2 (The R foundation, 2020). At

Injected human tumor cells remain in fly hosts and affect hosts' lifespan. Ectopic transplantation
of tumorigenic tissues and cells such as Ras V12 /scrib −/− -expressing eye discs and Ras V12 -expressing primary cell lines into adult flies have been reported to undergo over-proliferation and promote the death of hosts 10,15,16 . To determine the behavior of human tumor cells in the fly body, we injected fluorescent-tagged cultured human tumor cells www.nature.com/scientificreports/ into flies' abdomens using the established allograft protocol with modifications (see the "Materials and methods" for details) 8,10 . We used the following human tumor cell lines for xenografting: (1) HeLa-Kyoto (human cervical carcinoma cells), (2) A549 (lung epithelial adenocarcinoma cells), (3) MCF7 (breast cancer cells), and (4) DLD-1 (colorectal adenocarcinoma cells) (see "Materials and methods" for the specific cell names and culturing methods). After transplantations of human tumor cells into host flies, we monitored the fluorescence of tumor cells in the abdomen through fluorescence microscopy to evaluate tumor spreading and examined the survival rate of host flies (Fig. 1a), but no fluorescence was observed when PBS alone was injected (Fig. 1b [row 1]). Throughout our observations, the fluorescence of HeLa cells and MCF-7 cells disappeared 3-5 days after injection (Fig. 1b [rows 2 and 3], c,d,g). In contrast, the fluorescence of A549 cells and DLD-1 cells persisted for 10-13 days (Fig. 1b [rows 4 and 5], e-g). In addition to the duration of fluorescent signals, we observed differences in the patterning of the tumor fluorescence: A549 cells spread and persisted in broader areas in the abdomen while DLD-1 cells were frequently clustered, exhibiting spotty fluorescence within the host abdomen (Fig. S1). As a positive control for tumorigenic proliferation in host flies, we injected Ras V12 /scrib −/− cells and observed the gradual spread of Ras V12 /scrib −/− cells over 7 days in the host abdomen, which was associated with extremely low survival rate (Fig. 1b [row 6], h). While xenograft flies with human tumor cells survived longer than the Ras V12 /scrib −/− -transplanted flies, xenograft flies died significantly earlier than the PBS-injected control flies, except for those transplanted with HeLa cells (Fig. 1h).
Overall, human tumor cells were able to survive for several days in the host flies until they were cleared out ( Fig. 1b-g). In contrast, Drosophila Ras V12 /scrib −/− cell-transplanted flies exhibited expansion of the fluorescence (Fig. 1b [row 6]). One potential factor influencing this decreased viability of human tumor cells in fly bodies was the optimal temperature difference between human cells and fly cells. The reported optimal temperature for healthy Drosophila melanogaster is between 14-29 °C, with a common incubation temperature of 25 °C in laboratories 17,18 . By contrast, mammalian cells are commonly cultured at a much higher temperature of 37 °C and likely experience cellular stress when cultured at lower temperatures 19 . We thus chose 29 °C for the incubation of fly hosts as an intermediate temperature between the optimum temperatures for flies and humans. www.nature.com/scientificreports/  www.nature.com/scientificreports/ To test whether human tumor cell lines exhibit growth at this intermediate temperature, we cultured these cell lines in vitro at 29 °C. As expected, human tumor cell lines grew exponentially at different rates at 37 °C, with HeLa and A549 cells having the best growth rates (Fig. 1i). By contrast, we found a decreased growth rate of most tumor cell lines when incubated at 29 °C (Fig. 1j). While HeLa cells showed a decrease in numbers over time at 29 °C, A549 cells still maintained proliferation, albeit at a slower rate (Fig. 1j). These results suggest that different cell lines have distinct growth rates at 37 °C and 29 °C, indicating differences in their capabilities to adapt to lower temperatures.
Based on xenograft and in vitro culture experiments, we observed a correlation between the temperature resistance of the cell lines and their behavior following transplantation into fly hosts: (1) A549 cells, which continue to be sufficiently proliferative when cultured at 29 °C, showed persistence of GFP fluorescence up to around 13 days and induced a lower survival rate in host flies after transplantation; and (2) HeLa cells, which have lower resistance to low temperatures than the other cells, showed an inability to survive for longer than 3 days. Given that both HeLa and MCF-7 cells disappeared from Drosophila bodies within 5 days despite their continued survival at 29 °C in vitro, we considered a possibility that additional factors contribute to the removal of human tumor cells from fly hosts.
Transplanted human lung cancer cells are clustered adjacent to fly host tissues. Our lifespan measurement results suggest that the presence and behavior of human tumor cells detrimentally affect the systems of host flies (Fig. 1b-h). To investigate the characteristics and potential interactions between human tumor cells and fly tissues at the cellular level, we observed the fluorescently labeled human tumor cells in detail in host flies at 3 days after transplantation.
Prior to fluorescent staining for host flies, we washed our samples with PBS to remove cells suspended in the hemolymph. Following fixation and staining, we imaged the samples using confocal laser microscopy. Of note, we chose A549 cells for the following observations due to their longer persistence in fly hosts than other cell lines. In addition to the A549 cells, we also performed allografts and observed two Drosophila tumor models-Ras V12 (Fig. 2a) and Ras V12 /scrib −/− (Fig. 2b,b′)-as negative and positive controls in terms of tumorigenicity, respectively 10 . As expected, Ras V12 /scrib −/− cells grew better and formed bigger clusters than Ras V12 benign tumor (Figs. 1b, 2b,b′).
After transplantation, we confirmed the presence of A549 cells within the host's tissues sporadically located adjacent to fly host tissues (Fig. 2c,c′,d). In addition, some of the cells formed well-defined clusters within the muscle layer of the gut (Fig. 2e,f-1,f-2). These observations revealed that A549 cells, despite a different origin with a reduced proliferation rate at 29 °C compared to 37 °C, still displayed clustering behaviors, similar to Ras V12 /scrib −/− cells. Together with the lifespan measurements, these results suggest that other factors play roles in preventing the survival of A549 cells in host flies.

Drosophila innate immunity suppresses transplanted human tumor cell survival. It is widely
known that Drosophila, like many insects, only possesses an innate immune system 12 . Drosophila innate immunity consists of two pathways: the immune deficiency (Imd) and Toll pathway, both of which induce the production of antibacterial and antifungal peptides, including Drosomycin and Diptericin to fight against bacterial or fungal infection. However, how the immune system responds to the cells of different species, particularly from more phylogenetically distant organisms, such as human cells, is still unknown.
Our finding that A549 cells are visually adjacent to host tissues 3 days post-injection while gradually disappearing over time suggested a possibility that the Drosophila innate immunity may come into play in rejecting the human tumor cells. To test this possibility, we utilized the Imd pathway component mutants PGRP-LC Δ5 , Dredd D44 and Rel E20 for which viable homozygous lines are readily available and widely used. After transplantation of A549 cells into these mutant flies, we examined the impact of innate immunity on the growth of A549 cells and found that the transplanted A549 cells significantly survived longer in the immunodeficient flies-on average 14-16 days for PGRP-LC Δ5 and Dredd D44 hosts and until death for Rel E20 hosts, compared with only 10 days average in control w 1118 flies (Fig. 3a-f). Note that the difference in persistence of the A549 cells fluorescence in w 1118 host flies in Fig. 3a, compared with the ones in Fig. 1b, may be due to the number of cells injected (see "Materials and methods"). In addition, the transplanted A549 cells seemed to be more widespread with stronger fluorescence in the immunodeficient flies than in the w 1118 flies (Fig. 3a). Similar to the injections in w 1118 flies, the location of fluorescence was consistent over time for A549 cells in the immunodeficient fly hosts (Fig. 3a,  rows 2-4). These observations together suggest that A549 cells can survive longer in flies without a functional innate immune system. We next investigated the effects of A549 cells when injected into immunodeficient hosts by measuring the hosts' lifespan. We observed significantly earlier mortality soon after A549 cell injection (Fig. 3g,h). While we consider the existence of A549 cells to be the main cause of the host fly death, it may also be due to the stress associated with injection. To evaluate this possibility, we measured the lifespan of non-injected and PBS injected flies and compared the findings with the A549 cell-injected flies. As a result, we observed that PBS injections caused a minor, but significant increase in mortality of PGRP-LC Δ5 flies compared to non-injected flies, but not in Dredd D44 (Fig. 3g,h), suggesting that injection stress can cause a minor effect on fly host survival. Given that injections with A549 cells cause significant reduction in survival rate compared to controls, human tumor cells likely detrimentally affect fly hosts. Altogether, our results suggest that transplanted human tumor cells are able to survive and cause negative effects on Drosophila hosts, and that the fly host's immune system is a significant impeding factor for the growth of xenografted cells. www.nature.com/scientificreports/

Discussion
In this study, we performed xenograft experiments using different human tumor cell lines and explored the impacts of transplanted tumor cells in the host flies. We identified two factors that could influence the behavior and survival of the transplanted human tumor cells in Drosophila: the incubation temperature and the host immune system. The lower growth rate of human tumor cells in Drosophila hosts can be explained by both the optimal temperature and phylogenetic and physiological differences between humans and Drosophila. The successes of xenografting in zebrafish at relatively low temperatures (28 °C) [20][21][22] showed some promise for transplantation in Drosophila melanogaster if incubated at temperatures above 28 °C, or by developing a model in more heat-resistant Drosophila species. Indeed, several Drosophila species in the American Sonoran Desert such as Drosophila mojavensis exhibit the temperature resistance and are capable of mating at temperatures up to the mid-30s °C, with a thermal tolerance of up to around 40 °C 23,24 . Utilizing a more heat-tolerant species of Drosophila might increase our likelihood of establishing a viable invertebrate xenograft model. The other important factor is the difference between the immune system of vertebrates and invertebrates like insects including Drosophila. While vertebrate models such as mice and zebrafish have both acquired and innate immunity, Drosophila possess only innate immunity 12 . We showed that the loss of innate immunity in Imd mutant flies extended the survival of A549 cells post-injection and further reduced the host lifespan (Fig. 3), indicating the role of innate immunity to repel cells originating from more complex organisms and cancer cells, not only bacteria or fungi. The two innate host defense systems in Drosophila, Toll and Imd pathways, are homologous to NF-κB signaling in mammals 25 . In this early attempt, we opted to investigate the role of innate immunity using Imd pathway mutants. The Imd pathway contains components which share homologies with mammalian TNF signaling, and in particular, they are functionally homologous to the mammalian TNFR signaling pathway with the activity of Drosophila NF-κB homolog relish (rel) 26,27 . NF-κB activation in mammals involves the phosphorylation of, and subsequent degradation of the inhibiting IκB protein 28 . In Drosophila, Rel contains the IκB domain at the C-terminal, and requires cleavage by the caspase Dredd to split Rel into Rel-49 and Rel-68 [29][30][31] . Ultimately the cleaved Rel-68 translocates into the nucleus and binds to and activates the promoters of the antimicrobial peptide genes, similar to the activated NF-κB 27 . The mammalian TNFR signaling also controls transcription of several mammalian antimicrobial peptides (AMPs), including β-defensins, and recent studies have also described the potential utility of AMPs as anticancer agents 32,33 . Similarly, the Drosophila dlg-induced tumor is sensitive to the Drosophila homologue Defensin, mediated by TNF signaling 34 . With many similarities to TNFR signaling between vertebrates and fly, the Drosophila model provides an opportunity to study the activation and responses of the innate immune system exclusively, triggered by the introduction of non-microbial foreign cells. The detailed mechanisms underlying how Drosophila innate immunity affects the growth and survival of transplanted human tumor cells will need to be clarified. It will be also interesting to explore the capabilities of Drosophila Defensins against mammalian tumor cells.
Our main goal was to take the first step toward the establishment of a new PDX model using invertebrate Drosophila. Current xenograft attempts have been conducted only in vertebrate models, such as mice and zebrafish, due to their evolutionarily conserved similarities to the human body and cellular structures. Compared to the long lifespan of these vertebrate models, Drosophila have a relatively short lifespan, which makes it simpler to establish the experimental duration and is potentially useful for screening drug safety and toxicity, as we are able to measure the host lifespan after injection. Indeed, with the goal of establishing a "personalized Drosophila model", Bangi et al. developed a pipeline where a colon cancer patient's genomic profile was analyzed, and the specific mutations were introduced in Drosophila 35 . Large-scale drug screening was then conducted in order to select the most effective candidate drug combinations 35 . In contrast, we aim to approach personalized medicine differently, as we have performed direct transplantation of the donor cells that carry the mutation profile of the original donor, not by expressing Drosophila orthologs. While we showed that human tumor cellstransplanted flies died earlier than controls, one caveat is that the hosts' deaths occurred later than the removal of visible fluorescent signals. This leads to a question of whether the death was directly caused by the presence of human tumor cells or due to irreversible damage induced by the transplanted cells. It is also possible that transplanted tumor cells irreversibly influence host tissues via tumor-host interactions, and this effect leads to lethality. Future studies should focus on the detailed mechanisms of tumor-host interactions from transplantation to host death on a time scale.
In summary, this report showcased the first observations of the behavior of mammalian cell lines when transplanted into Drosophila and revealed that temperature and host innate immunity influence the compatibility between mammalian donor cells and Drosophila hosts. Further studies will be needed to confirm the value of this model as a novel PDX model as well as future drug screening platforms for cancer research. www.nature.com/scientificreports/